Method for the treatment of amyloidoses

ABSTRACT

The present invention relates to a method for the treatment of an amyloidosis such as Alzheimer&#39;s disease in a subject in need thereof, characterized in that it comprises administering an agonist of the P/Q type voltage-gated presynaptic calcium channel to said subject.

CROSS-REFERENCE

This application is the National Stage of International Application No.PCT/EP2008/001549, filed on Feb. 27, 2008, which claims the benefit ofEuropean Application Serial No. 07020257.7, filed Oct. 16, 2007,European Application Serial No. 08000325.4, filed Jan. 9, 2008, and U.S.Provisional Application Ser. No. 60/903,700, filed Feb. 27, 2007, all ofwhich are incorporated herein by reference in its entirety.

The present invention relates to a method for the treatment of anamyloidosis such as Alzheimer's disease.

Alzheimer's disease (AD), the most frequent cause for dementia among theaged with an incidence of about 10% of the population above 65 years, isa dementing disorder characterized by a progressive loss of cognitiveabilities and by characteristic neuropathological features comprisingextracellular amyloid deposits, intracellular neurofibrillary tanglesand neuronal loss in several brain regions (Mattson, M. P. Pathwaystowards and away from Alzheimer's disease. Nature 430, 631-639 (2004);Hardy, J. & Selkoe, D. J. The amyloid hypothesis of Alzheimer's disease:progress and problems on the road to therapeutics. Science 297, 353-356(2002)). The principal constituents of the amyloid deposits are amyloidβ peptides (Aβ) which arise from the (β-amyloid precursor protein (APP)by proteolytic cleavage.

Both cerebral amyloid deposits and cognitive impairments very similar tothose observed in Alzheimer's disease are also hallmarks of Down'ssyndrome (trisomy 21), which occurs at a frequency of about 1 in 800births. Hence, Alzheimer's disease and Down's syndrome are jointlytermed “amyloidoses”.

Recently, however, it was shown that in amyloidoses soluble, globular Aβoligomers (hereinafter referred to as Aβ globulomers), rather than theeponymous insoluble amyloid deposits, are the causative agents for theimpairment of higher-level functions, such as memory function, asindicated by its suppressing effect on long-term potentiation(WO2004/067561; Barghorn S. et al., J. Neurochem. 95: 834-847 (2005);WO2006/094724).

The term “Aβ globulomer” here refers to a particular soluble, globular,non-covalent association of Aβ peptides, possessing homogeneity anddistinct physical characteristics. Aβ globulomers are stable,non-fibrillar, oligomeric assemblies of Aβ peptides which are obtainableby incubation with anionic detergents, in particular as described inWO2004/067561. In contrast to Aβ monomer and fibrils, these globulomersare characterized by defined assembly numbers of subunits(WO2004/067561). The globulomers have a characteristic three-dimensionalglobular type structure (“molten globule”, see Barghorn et al., J.Neurochem. 95: 834-847 (2005)). They have been shown to closely mimicthe properties, behaviour and effects of naturally occurring soluble Aβoligomers.

Soluble Aβ oligomer was found to impair the functioning of the centralnervous system even before the onset of cytotoxicity. However, the exactmechanisms whereby soluble Aβ oligomer causes memory failure inamyloidoses has not been elucidated so far, and a lack of understandingof such mechanisms has so far hampered the development of rationaltherapeutic approaches for inhibiting the further progression of thedisease or compensating the damage already done.

It was thus an object of the present invention to provide a new approachto the treatment of amyloidoses such as Alzheimer's disease, inparticular to rehabilitating treatment such as the restoration ofcognitive abilities in amyloidoses such as Alzheimer's disease.

Surprisingly, it was now found that Aβ globulomer exerts its detrimentaleffects essentially by hampering normal ion fluxes through the P/Q typepresynaptic calcium channel, reducing presynaptic neurotransmitterrelease and inhibiting spontaneous synaptic activity and therebyinterfering with the proper functioning of the central nervous systemeven before the onset of manifest neural cytotoxicity, and thatactivation of the P/Q type presynaptic calcium channel is thereforeeffective in compensating these effects (increasing the extracellularCa²⁺ concentration (P/Q type presynaptic calcium channel agonism) waseffective in reversing the inhibitory effect of Aβ globulomer on the P/Qtype voltage-gated presynaptic calcium chnnel).

The present invention thus relates to a method for the treatment of anamyloidosis, preferably Alzheimer's disease, in a subject in needthereof, comprising administering an agonist of the P/Q typevoltage-gated presynaptic calcium channel to said subject.

The P/Q type voltage-gated presynaptic calcium channel (the channel isalso referred to as Ca_(v)2.1 channel and the associated currents as P/Qtype currents) belongs to the group of voltage-gated calcium channelswhich mediate the influx of calcium ions into excitable cells. Theopening state of a voltage-gated channel is controlled by the electricalstate of the surrounding membrane; however, the responsiveness of theP/Q type voltage-gated presynaptic calcium channel to membranedepolarization is extensively modulated, both qualitatively andquantitatively, by and/or through its interaction partners.

As used herein, a “P/Q type voltage-gated presynaptic calcium channel”is a voltage-gated calcium channel that is functionally characterized byits sensitivity towards ω-agatoxin IVA (a well-known funnel web spidervenom).

According to a particular embodiment, ω-agatoxin IVA acts as a gatingmodifier of the P/Q type voltage-gated presynaptic calcium channel(e.g., P type Kd=1-3 nM; Q type Kd=100-200 nM). Further, P/Q typevoltage-gated presynaptic calcium channels according to the presentinvention may be characterized by one or more than one of the followingfeatures:

-   -   (i) requires strong depolarization for activation (high-voltage        activation); and    -   (ii) no or slow inactivation.

The P/Q type voltage-gated presynaptic calcium channel according to thepresent invention comprises an α1 subunit. According to a particularembodiment of the invention, the α1 subunit has an amino acid sequencewith at least 70%, advantageously at least 80%, preferably at least 90%,more preferably at least 95% and in particular at least 98%, e. g. atleast 99%, amino acid sequence identity with the sequence SEQ ID NO:1.The α1 subunit incorporates the conduction pore, the voltage sensor andgating apparatus, and sites of channel regulation by second messengers,drugs, and toxins.

Usually, the P/Q type voltage-gated presynaptic calcium channel alsocomprises an α2-δ subunit and a β subunit. It may also comprise an γsubunit. In a particular embodiment of the invention, the α2-δ subunit,when present, has at least 70%, advantageously at least 80%, preferablyat least 90%, more preferably at least 95% and in particular at least98%, e. g. at least 99%, amino acid sequence identity with the sequenceSEQ ID NO:2. In a further particular embodiment of the invention, the βsubunit, when present, has at least 70%, advantageously at least 80%,preferably at least 90%, more preferably at least 95% and in particularat least 98%, e. g. at least 99%, amino acid sequence identity with thesequence SEQ ID NO:3.

Further characteristic features of P/Q type voltage-gated presynapticcalcium channels are described in Catterall W A, Perez-Reyes E, Snutch TP, Striessnig J. International Union of Pharmacology. XLVIII.Nomenclature and structure-function relationships of voltage-gatedcalcium channels. Pharmacol Rev. 57: 411-25 (2005), which is hereinincorporated by reference in its entirety.

As used herein, an “agonist of the P/Q type voltage-gated presynapticcalcium channel” is any substance that increases the flow of calciumions through the P/Q type voltage-gated presynaptic calcium channel.

According to a particular embodiment, the agonist of the inventionincreases the open probability of the channel.

According to a further particular embodiment, the agonist of theinvention directly interacts with the closed channel to open it.

According to a further particular embodiment, the agonist of theinvention increases the duration of the open state once the channel hasbeen opened.

According to a further particular embodiment, the agonist of theinvention interacts with the time constants of voltage-gated activation,voltage-gated inactivation and voltage-gated deinactivation in a waywhich results in an increased net calcium flux under physiologicalconditions and which may be voltage-dependent in itself.

According to a further particular embodiment, the agonist of theinvention changes one or more transition probabilities between thedifferent states of the channel (closed, open, inactivated).

Hence an agonist of the P/Q type voltage-gated presynaptic calciumchannel will increase ion flux through the P/Q type voltage-gatedpresynaptic calcium channel.

The agonist of the P/Q type voltage-gated presynaptic calcium channel ispreferably a partial or complete agonist of the P/Q type voltage-gatedpresynaptic calcium channel, and more preferably a complete agonist ofthe P/Q type voltage-gated presynaptic calcium channel.

As used herein, a “partial agonist of the P/Q type voltage-gatedpresynaptic calcium channel” is an agonist which is not capable even athigh concentration of causing the maximal activating effect of the P/Qtype voltage-gated presynaptic calcium channel, as defined above. In aparticular embodiment a partial agonist does not lead to maximal calciumflow through the P/Q type voltage-gated presynaptic calcium channel atsaturating concentration.

As used herein, a “complete agonist of the P/Q type voltage-gatedpresynaptic calcium channel” is an agonist which is capable of inducing,at suitable concentrations, maximal activation of the P/Q typevoltage-gated presynaptic calcium channel, as defined above.

The person skilled in the art will understand that an agonist of the P/Qtype voltage-gated presynaptic calcium channel may either bind directlyto the P/Q type voltage-gated presynaptic calcium channel, i. e. bybinding the calcium channel molecule, e. g. by forming a covalent ornon-covalent attachment to said calcium channel molecule, or exert itseffect on the ion channel predominantly without direct physical contactbetween the agonist and said calcium channel, the effect being mediated,in this case, by any of a wide range of go-betweens. These include butare not limited to molecules with a capacity of complexing said calciumchannel that is modified in the presence of the agonist; catalyticallyactive molecules which regulate said calcium channel in a way that isinfluenced by the presence of the agonist; and physico-chemical effects,in particular membrane effects, subject to influences of the agonist,such as shifts in the molecular arrangement, fluidity, conductivity,etc. of the membrane. In particular, an agonist of the P/Q typevoltage-gated presynaptic calcium channel may bind to both the P/Q typevoltage-gated presynaptic calcium channel and components of itsenvironment, thereby influencing the mutual interaction. In all of thesecases, under otherwise identical conditions the behaviour of saidcalcium channel in the presence of an effective amount of the agonistwill be detectably different from that in the absence of said agonist.

In the context of the present invention, the term “bind” is usedgenerically to denote any immediate association between two molecules,which may be covalent or non-covalent, thus including covalent bonds,hydrogen bridges, ionic interactions, hydrophobic associations, van derWaals forces, etc. It will thus be understood that the term also extendsto the temporary association of a first molecule with a catalyticallyactive second molecule, wherein said second molecule performsmodifications on said first molecule which, and consequently whoseeffects, outlast the actual contact between said first and said secondmolecule, e. g. generation or removal of covalent bonds.

In a particular embodiment of the invention, the agonist increases netcalcium flux through the P/Q type voltage-gated presynaptic calciumchannel with an EC50 of less than 120 μM, preferably of less than 10 μM,and in particular of less than 1 μM. Here the EC50 is the agonistconcentration required for obtaining 50% of a maximum effect of thisagonist determined using the patch-clamp method for whole-cell recordingof channel activity.

The standard method employed here for all determinations of Ca⁺⁺currents is a patch-clamp method using 120 mM NMG.Cl, 10 mM TEA.Cl, 14mM creatine phosphate, 6 mM MgCl₂, 1.mM CaCl₂ 10 mM NMG.HEPES, 5 mMTris₂.ATP and 11 NMG₂.EGTA as internal, and 30 mM BaCl₂, 100 mM NMG.Cl,10 mM NMG.HEPES and 15 mM glucose as external solution, both adjusted toa pH of about 7.2-7.3, for measuring stably transfected BHK (BabyHamster Kidney) cells expressing the al component together with the α2δand βIB components of the P/Q type voltage-gated presynaptic calciumchannel.

Further details of said standard method have been described by ZafirBuraei et al., Roscovitine differentially affects CaV2 and Kv channelsby binding to the open state, Neuropharmacology (2006),doi:10.1016/j.neuropharm.2006.10.006 (corresponds to issue 52, 2007,pages 883-894), which is herein incorporated by reference in itsentirety.

Unexpectedly, it was further found that other presynaptic calciumchannels, such as the N and R type voltage-gated presynaptic calciumchannels, are not susceptible to Aβ, in particular to Aβ globulomer, andit is known to the skilled person that these types of channels may allbe present in parallel at any given synapse, or one of them may bedominant in terms of abundance and thus account for the major part ofthe Ca⁺⁺ influx. It is thus, in the interest of reducing side effects,advantageous to employ in the method of the present invention an agonistwhich specifically affects the P/Q type voltage-gated presynapticcalcium channel only. Thus, it is advantageous if the agonist affectsthe P/Q type voltage-gated presynaptic calcium channel with lower EC50than either the N type or the R type, or both the N and the R typevoltage-gated presynaptic calcium channels. In a particular embodimentof the invention, the agonist of the P/Q type voltage-gated presynapticcalcium channel thus increases net calcium flux through the L typevoltage-gated presynaptic calcium channel with an EC50 of more than 54μM, preferably of more than 100 μM, and in particular of more than 1000μM, using the standard method as defined above. In a further particularembodiment of the invention, it increases net calcium flux through the Ntype voltage-gated presynaptic calcium channel with an EC50 of more than54 μM, preferably of more than 100 μM, and in particular of more than1000 μM, using the standard method as defined above. In a furtherparticular embodiment of the invention, it increases net calcium fluxthrough the R type voltage-gated presynaptic calcium channel with anEC50 of more than 54 μM, preferably of more than 100 μM, and inparticular of more than 1000 μM, using the standard method as definedabove.

It has been reported that inhibition of CDKs (cyclin-dependant kinases),which kinases are known to be crucial elements of the cell cycle,inhibits dedifferentiation and proliferation and thereby stimulatesneurons to increase their expression of ion channels such asvoltage-gated presynaptic calcium channels. However, due to thenaturally highly pleiotropic effects of CDK inhibitors and inparticularly their propensity to induce apoptosis, which is expected tooutweigh the benefits due to the neurotoxicity caused thereby, it ispreferred that the agonists for use in the methods of the presentinvention do not exert any significant inhibitory effect on CDKs such asCDK2 (cyclin A) and CDK5 (p35). In a particular embodiment of theinvention, the agonist thus has an IC50 for CDK5, in particular forhuman CDK5, of more than 0.2 μM, preferably of more than 10 μM, and inparticular of more than 100 μM. In a further particular embodiment ofthe invention, the agonist thus has an IC50 for CDK2, in particular forhuman CDK2, of more than 0.7 μM, preferably of more than 10 μM, and inparticular of more than 100 μM. Preferably, the agonist has an IC50 forany CDK, in particular for any human CDK, of more than 0.5 μM,preferably of more than 10 μM, and in particular of more than 100 μM.These IC50 values all refer to the activity of the respective CDK/cyclincomplex in a radioactive kinase assay using 15 μM of [γ-³²P]ATP asphosphate donor and an appropriate phosphorylation target protein (1mg/ml histone H1 or retinoblastoma protein complexed withglutathione-S-transferase, respectively) as acceptor in a reactionbuffer comprising 60 mM glycerol-2-phosphate, 15 mM p-nitrophenylphosphate, 25 mM MOPS pH 7.2, 5 mM EGTA, 15 mM MgCl₂ and 1 mMdithiotreitol, as described by Meijer et al., Eur. J. Biochem. 234:527-536 (1997).

The metabolism of APP and its products such as Aβ is complex and not yetfully understood. Therefore, in a particular embodiment of theinvention, the agonist as such does not after APP expression, Aβformation or processing, e. g. soluble Aβ oligomer or Aβ fibrilformation, in the central nervous system. In another particularembodiment, however, its use may be combined with a suitable treatment,e. g. a treatment to suppress the formation of soluble Aβ oligomersand/or to promote their degradation and/or elimination from the centralnervous system; essentially any such treatment may be combined with themethods of the present invention.

Roscovitine has been described as an agonist of the P/Q typevoltage-gated presynaptic calcium channel (Yan Z, Chi P, Bibb J A, RyanT A and Greengard P., J. Physiol. 540 : 761-770 (2002)). As used herein,the term “roscovitine” denotes the compound(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine and any ofits isomers, in particular2-(R)-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine.

However, roscovitine is known to reduce APP formation by binding to andinhibiting the action of human CDKs, in particular CDK5, thereby beingprone to the adverse effects outlined above. Therefore, agonists otherthan roscovitine or any pharmacologically useful salt or derivative ofroscovitine, in particular such as may be readily converted toroscovitine in vivo (prodrug), are preferred.

According to a particular embodiment, suitable agonists of the P/Q typevoltage-gated presynaptic calcium channel of the invention are selectedamong the roscovitine analogues of formula (Ia):

wherein

-   -   R¹ is hydrogen or C₁-C₆-alkyl;    -   R^(2a), R^(2b) are independently hydrogen, C₁-C₆-alkyl,        C₂-C₆-alkenyl, C₃-C₈-cycloalkyl, optionally substituted        C₆-C₁₂-aryl or optionally substituted C₆-C₁₂-aryl-C₁-C₄-alkyl,        or    -   R^(2a), R^(2b) together are C₂-C₅-alkylene;    -   Q is NR³;    -   R³ is hydrogen, C₁-C₆-alkyl or optionally substituted        C₆-C₁₂-aryl;    -   X is N or CR⁴;    -   R⁴ is hydrogen or C₁-C₆-alkyl,    -   Y is N or CR⁵; and    -   R⁵ is hydrogen or C₁-C₆-alkyl,        and the pharmacologically useful salts thereof.

According to a further particular embodiment, suitable agonists of theP/Q type voltage-gated presynaptic calcium channel of the invention areselected among the roscovitine analogues of formula (Ib):

wherein

-   -   R¹ is hydrogen or C₁-C₆-alkyl;    -   R^(2a), R^(2b) are independently hydrogen, C₁-C₆-alkyl,        C₂-C₆-alkenyl, C₃-C₈-cycloalkyl, optionally substituted        C₆-C₁₂-aryl or optionally substituted C₆-C₁₂-aryl-C₁-C₄-alkyl,        or    -   R^(2a), R^(2b) together are C₂-C₅-alkylene;    -   Q is NR³;    -   R³ is hydrogen, C₁-C₆-alkyl or optionally substituted        C₆-C₁₂-aryl;    -   X is N or CR⁴;    -   R⁴ is hydrogen or C₁-C₆-alkyl,    -   Y is N or CR⁵; and    -   R⁵ is hydrogen or C₁-C₆-alkyl,        and the pharmacologically useful salts thereof.

According to a further particular embodiment, suitable agonists of theP/Q type voltage-gated presynaptic calcium channel of the invention areselected among isoproterenol and isoproterenol analogues of formula(II):

wherein

-   -   R¹ is C₁-C₆-alkyl or C₃-C₈-cycloalkyl;    -   R^(2a), R^(2b), R^(2c), R^(2d), R^(2e) are independently        hydrogen, halogen, C₁-C₄-alkyl, optionally substituted phenyl,        OH, SH, CN, CF₃, O—CF₃, C₁-C₄-alkoxy, NH₂, NH—C₁-C₄-alkyl,        N—(C₁-C₄-alkyl)₂, or    -   R^(2b) and R^(2c) or R^(2b) and R^(2d) together with the carbon        atoms to which they are attached form an optionally substituted        anellated C₅-C₇ carbocyclic ring;    -   and the pharmacologically useful salts thereof.

Provided that the compounds of the formulae (Ia), (Ib) and (II) exist indifferent spatial arrangements, for example if they possess one or morecenters of asymmetry, polysubstituted rings or double bonds, or asdifferent tautomers, it is also possible to use enantiomeric mixtures,in particular racemates, diastereomeric mixtures and tautomericmixtures, preferably, however, the respective essentially pureenantiomers, diastereomers and tautomers of the compounds of formulae(Ia), (Ib) and (II) and/or of their salts.

The pharmacologically useful salts of the compounds of the formulae(Ia), (Ib) and (II) are especially acid addition salts withphysiologically tolerated acids. Examples of suitable physiologicallytolerated organic and inorganic acids are hydrochloric acid, hydrobromicacid, phosphoric acid, sulfuric acid, C₁-C₄-alkylsulfonic acids, such asmethanesulfonic acid, cycloaliphatic sulfonic acids, such asS-(+)-10-campher sulfonic acid, aromatic sulfonic acids, such asbenzenesulfonic acid and toluenesulfonic acid, di- and tricarboxylicacids and hydroxycarboxylic acids having 2 to 10 carbon atoms, such asoxalic acid, malonic acid, maleic acid, fumaric acid, lactic acid,tartaric acid, citric acid, glycolic acid, adipic acid and benzoic acid.Other utilizable acids are described, e.g., in Fortschritte derArzneimittelforschung [Advances in drug research], Volume 10, pages 224ff., Birkhauser Verlag, Basel and Stuttgart, 1966.

The organic moieties mentioned in the above definitions of the variablesare—like the term halogen—collective terms for individual listings ofthe individual group members. The prefix C_(n)-C_(m) indicates in eachcase the possible number of carbon atoms in the group.

Unless specified, the term “substituted” means that a radical may besubstituted with 1, 2 or 3, especially 1 or 2, substituent selected fromthe group consisting of halogen, C₁-C₄-alkyl, OH, SH, CN, CF₃, O—CF₃,C₁-C₄-alkoxy, NH—C₁-C₄-alkyl, N—(C₁-C₄-alkyl)₂, in particular with 1, 2oder 3 substituents selected from the group consisting of halogen,methyl, OH, CN, CF₃, O—CF₃, methoxy, NH₂, NH—CH₃, and N—(CH₃)₂.

The term halogen denotes in each case fluorine, bromine, chlorine oriodine, in particular fluorine or chlorine.

C₁-C₄-Alkyl is a straight-chain or branched alkyl group having from 1 to4 carbon atoms. Examples of an alkyl group are methyl, C₂-C₄-alkyl suchas ethyl, n-propyl, iso-propyl, n-butyl, 2-butyl, iso-butyl ortert-butyl. C₁-C₂-Alkyl is methyl or ethyl, C₁-C₃-alkyl is additionallyn-propyl or isopropyl.

C₁-C₆-Alkyl is a straight-chain or branched alkyl group having from 1 to6 carbon atoms. Examples include methyl, C₂-C₄-alkyl as mentioned hereinand also pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl,2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl,1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl,2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl,1-ethyl-1-methylpropyl and 1-ethyl-2-methylpropyl.

C₃-C₈-Cycloalkyl is a cycloaliphatic radical having from 3 to 12 carbonatoms. In particular, 3 to 6 carbon atoms form the cyclic structure,such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The cyclicstructure may be unsubstituted or may carry 1, 2, 3 or 4 C₁-C₄ alkylradicals, preferably one or more methyl radicals.

C₆-C₁₂-aryl-C₁-C₄-alkyl is a straight-chain or branched alkyl grouphaving 1 to 4 carbon atoms, preferably 1 to 3 carbon atoms, morepreferably 1 or 2 carbon atoms, in particular 1 or two carbon atoms,wherein one hydrogen atom is replaced by C₆-C₁₂-aryl, such as in benzyl.

C₂-C₆-Alkenyl is a singly unsaturated hydrocarbon radical having 2, 3,4, 5 or 6 carbon atoms, e.g. vinyl, allyl (2-propen-1-yl),1-propen-1-yl, 2-propen-2-yl, methallyl(2-methylprop-2-en-1-yl) and thelike. C₃-C₄-Alkenyl is, in particular, allyl, 1-methylprop-2-en-1-yl,2-buten-1-yl, 3-buten-1-yl, methallyl, 2-penten-1-yl, 3-penten-1-yl,4-penten-1-yl, 1-methylbut-2-en-1-yl or 2-ethylprop-2-en-1-yl.

C₆-C₁₂-Aryl is a 6- to 12-membered, in particular 6- to 10-membered,aromatic cyclic radical. Examples include phenyl and naphthyl.

C₂-C₇-Alkylene is straight-chain or branched alkylene group having from2 to 7 carbon atoms. Examples include ethylene, 1,3-propylene,1,4-butylene and 1,5-pentylene.

Particular roscovitine analogues are defined as follows.

According to a particular embodiment, R¹ in formula (Ia) and formula(Ib) is hydrogen or C₁-C₃-alkyl. C₁-C₃-alkyl is in particular ethyl orisopropyl.

According to a further particular embodiment, R^(2a) and R^(2b) informula (Ia) and formula (Ib) are independently hydrogen, C₁-C₃-alkyl,in particular ethyl, C₂-C₃-alkenyl, in particular allyl,C₃-C₈-cycloalkyl, in particular cyclohexyl, optionally substitutedphenyl or optionally substituted benzyl. Substituted phenyl is inparticular phenyl substituted with 1, 2 or 3 substituent which areindependently selected from the group consisting of halogen, methyl,methoxy and NH₂. According to a further particular embodiment, R³ ishydrogen and R^(2b) is as defined. Alternatively, R^(2a) and R^(2b) informula (Ia) and formula (Ib) together are C₂-C₇-alkylene, in particular1,5-pentylene, and thus form a 3- to 8-membered, in particular6-membered, ring including the nitrogen atom to which they are attached.

According to a further particular embodiment, R³ in formula (Ia) andformula (Ib) is C₁-C₆-alkyl, in particular C₁-C₃-alkyl, or optionallysubstituted C₆-C₁₂-aryl, in particular optionally substituted phenyl.

According to a further particular embodiment of formula (Ia), X is N, Yis CR⁵ (in particular CH) and Q is NR³ (in particular N(C₁-C₃-alkyl),e.g. NCH₃, NCH₂CH₃ or NCH(CH₃)₂, or N(phenyl)); or X is CR⁴ (inparticular CH), Y is N and Q is NR³ (in particular N(C₁-C₃-alkyl) orN(phenyl)).

According to a further particular embodiment of formula (Ib), X is N, Yis CR⁵ (in particular CH) and Q is NR³ (in particular NCH₃).

Roscovitine analogues according to the present invention, in particular,include the following compounds

and their pharmacologically useful salts.

According to a particular embodiment, the roscovitine analogue is(1-ethyl-2-hydroxyethylamino)-6-(2-hydroxybenzyl)amino-9-isopropylpurine(hereinafter referred to as roscovitine analogue A). This roscovitineanalogue is capable of reversing the inhibitory effect of Aβ globulomeron synaptic transmission.

According to a particular embodiment, the agonists of the P/Q typevoltage-gated presynaptic calcium channel of formula (II) isisoproterenol. Isoproterenol has been described as an agonist of the P/Qtype voltage-gated presynaptic calcium channel (Huang C.-C., et al., TheJournal of Neuroscience, 1996, 16(3): 1026-1033, Huang C.-C., et al.,The Journal of Neuroscience, 1998, 18(6): 2276-2282). As used herein,the term “isoproterenol” (also known as isoprenaline) denotes4-[1-hydroxy-2-(1-methylethylamino)ethyl]benzene-1,2-diol. Isoproterenolis capable of reversing the inhibitory effect of Aβ globulomer onspontaneous synaptic activity, which is mediated by suppression of theP/Q type voltage-gated presynaptic calcium channel.

Particular isopreterenol analogues are defined as follows.

According to a further particular embodiment, R¹ in formula (II) isC₁-C₄-alkyl or C₃-C₆-cycloalkyl. C₁-C₄-alkyl is in particular methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl.C₃-C₆-cycloalkyl is in particular cyclopropyl or cyclohexyl.

According to a further particular embodiment, at least one, two or threeof R^(2a), R^(2b), R^(2c), R^(2d), R^(2e) in formula (II) is/aredifferent from hydrogen. In particular at least one of R^(2a), R^(2b),R^(2c), R^(2d) is different from hydrogen.

According to a further particular embodiment, R^(2a), R^(2b), R^(2c),R^(2d), are independently hydrogen, halogen, methyl, optionallysubstituted phenyl, OH, methoxy, CN, or NH₂, or R^(2b) and R^(2c) orR^(2c) and R^(2d) together with the carbon atoms to which they areattached form an optionally substituted anellated carbocyclic ring. Thecarbocyclic ring may be partially unsaturated (due to the benezenemoiety to which R^(2b), R^(2c) and R^(2d) are attached) or aromatic.According to a particular embodiment, R^(2b) and R^(2c) or R^(2c) andR^(2d) together with the benzene moiety to which they are attached forman optionally substituted 1,2,3,4-tetrahydronaphthalene or naphthalenemoiety.

Isopreterenol analogues according to the present invention, inparticular, include the following compounds

and their pharmacologically useful salts.

The compounds of the formula (Ia), (Ib) or (II) can be prepared byanalogy to methods which are well known in the art. Many of saidcompounds are commercially available.

Further agonists of the P/Q type voltage-gated presynaptic calciumchannel may be identified among compounds known per se by screening fortheir capacity to act as agonists of the P/Q type voltage-gatedpresynaptic calcium channel, preferably by screening using a methodcomprising determining the effect of a candidate compound on the openingstate of the P/Q type voltage-gated presynaptic calcium channel, mostconveniently by determining the effect of said compound on the Ca⁺⁺ fluxthrough the P/Q type voltage-gated presynaptic calcium channel. Suitablemeans for determining ion fluxes such as Ca⁺⁺ fluxes through the P/Qtype voltage-gated presynaptic calcium channel have been described inthe art (Yan Z, et al., 2002, supra; Buraei et al 2006, supra).

A method for determining whether any candidate compound is an agonist atthe P/Q type voltage-gated presynaptic calcium channel comprises thesteps of

-   -   (I) providing the P/Q type voltage-gated presynaptic calcium        channel; and    -   (II) determining Ca⁺⁺ fluxes through said P/Q type voltage-gated        presynaptic calcium channel in the presence and in the absence        of the candidate compound;        wherein an increase of the Ca⁺⁺ flux through the P/Q type        voltage-gated presynaptic calcium channel in the presence        relative to the Ca⁺⁺ flux through the P/Q type voltage-gated        presynaptic calcium channel in the absence of the candidate        compound is indicative of an agonistic effect of the candidate        compound at the P/Q type voltage-gated presynaptic calcium        channel. Alternatively, the agonistic effect can be assessed by        measuring Ba⁺⁺ currents through P/Q type voltage-gated        presynaptic calcium channels using methods well-known to the        skilled artisan.

The P/Q type voltage-gated presynaptic calcium channel is known per se(see, e. g., WO98/13490; Qian J and Noebels J L. J Neurosci 21:3721-3728, 2001; Yan Z, et al., 2002, supra). WO98/13490 in particulardiscloses the cDNA sequence for the human P/Q type voltage-gatedpresynaptic calcium channel, encoding a protein of 2261 amino acids.Methods for expressing a protein from a cDNA in vertebrate cells arewell-documented in the art; e. g. WO96/39512 discloses a process forgenerating cell lines expressing voltage-gated calcium channels. It isthus within the ken of the skilled person to provide the P/Q typevoltage-gated presynaptic calcium channel.

Expediently, the P/Q type voltage-gated presynaptic calcium channel isprovided on a living cell, which cell may be either in its naturalenvironment (in situ) or separated therefrom (ex vivo). In a particularembodiment, the cell to be used in the screening method is of a typethat naturally expresses the P/Q type voltage-gated presynaptic calciumchannel, e. g. a neuronal cell such as a hippocampal neuronal cell. Inanother embodiment, the cell to be used in the screening methodexpresses the P/Q type voltage-gated presynaptic calcium channel as aforeign gene. In this embodiment, it is preferred that the cellnaturally does not express any other voltage-gated presynaptic calciumchannels, e. g. a non-neural cell, e. g. a Xenopus oocyte. Conveniently,expression of the P/Q type voltage-gated presynaptic calcium channel inthe cells is verified using standard methology, e. g. by Northernblotting, RT-PCR, Western blotting, cytometry, binding of P/Q-specificligands such as ω-agatoxin, or pharmacological characterization, i. e.reduction of calcium current after agatoxin application.

In a further particular embodiment, said living cell further comprisesan agent for the in situ detection of calcium ion levels (i. e. acalcium sensor agent), e. g. a protein with a calcium-dependentluminescence or fluorescence, such as aequorin or cameleon (Putney P W.Calcium Signaling. CRC Press Inc, 2005). Such calcium sensor agents arewell-known to the skilled person, and essentially any of them may beused in the present invention. Without wishing to be bound by theory, itis believed that in suitable agents the conformation of the moleculechanges in a manner that depends on the local concentration of Ca⁺⁺,thereby hampering or facilitating physical processes, such as inter- orintramolecular energy transfers, that may be detected and correlatedwith calcium channel function by the experimentator. Thus, thefluorescence or luminescence of said calcium sensor agents is indicativeof the local (e. g. intracellular) calcium levels.

Hence, when the only functional calcium channel of the cell is the P/Qtype voltage-gated presynaptic calcium channel, increases inintracellular calcium concentrations

$\left( {\frac{\left\lbrack {Ca}^{++} \right\rbrack}{t} > 0} \right)$

indicate calcium fluxes through the P/Q type voltage-gated presynapticcalcium channel. Therefore, a rise in said increase

$\left( {{\frac{\left\lbrack {Ca}^{++} \right\rbrack_{C}}{t} > \frac{\left\lbrack {Ca}^{++} \right\rbrack_{0}}{t}},} \right.$

where [Ca⁺⁺]_(C) is the intracellular calcium concentration in the cellin the presence and [Ca⁺⁺]₀ in the absence of the candidate compound)indicates the P/Q agonist activity of a candidate substance and thus itspotential for the treatment of amyloidoses, as described above.

Suitable methods for the direct determination of ion fluxes, such as thevoltage-clamp method, are likewise known in the art (Sakmann B and NeherE. Single-Channel Recording. Springer US, 97 A.D.). Essentially,conductive microconnections with the inside and the outside of the cellmembrane are established, and the electrical reactivity of the systemunder different conditions is observed.

Preferably, prior to the measurement irrelevant ion channels are blockedusing inhibitors specific for said irrelevant channels (“pharmacologicalisolation” of the relevant channel or channels), eliminating thedependencies of the electrical status of the membrane on all channelsexcept the one or ones of interest (i. e. the P/Q channel). An activatorof the P/Q type voltage-gated presynaptic calcium channel and hence anagent suitable for the treatment of amyloidoses according to the presentinvention, as mentioned above, will thus be identified as an enhancer ofCa⁺⁺ flux when only the P/Q type voltage-gated presynaptic calciumchannel is expressed, or when all other calcium channels are blocked.

As all these methods for the determination of Ca⁺⁺ fluxes areessentially quantitative, they are also suitable for the identificationof an agonist with any particularly desired strength of agonistic effecton the P/Q type voltage-gated presynaptic calcium channel, wherein thestrength of the agonistic effect is the increase in calcium influxinduced by the agonist under the conditions selected.

Thus, an agent for the treatment of amyloidoses such as Alzheimer'sdisease can be identified by determining the effect of said agent on acell comprising at least the P/Q type voltage-gated presynaptic calciumchannel, in particular the effect on the Ca⁺⁺ flux through the P/Q typevoltage-gated presynaptic calcium channel of said living cell, whereinan agonist at the P/Q type voltage-gated presynaptic calcium channel ispotentially a suitable agent for the treatment of amyloidoses accordingto the present invention.

Among the agonists identified thereby, such as those having an affinityto the N type voltage-gated presynaptic calcium channel of less than anyparticularly desired value may be readily selected using methodsessentially known in the art, e. g. by employing the methods fordetection of Ca⁺⁺ fluxes disclosed above in combination with knownblockers for non-N type voltage-gated presynaptic calcium channels.

Suitable methods for determining affinity between molecules aregenerally well-known to the person skilled the art and comprise, withoutbeing limited to, determining radiation-free energy transfer,radiolabelling of ligands and co-immunoprecipitation. Likewise, theskilled person is familiar with suitable methods for determining theinhibitory effect of a compound on any given enzyme, and will thus beable to readily select among the agonist identified as described abovesuch as have an IC50 for any CDK, such as CDK5, of more than anyparticularly desired value.

As used herein, the term “administering” is used to denote delivering anagent to a subject, especially a human subject. Basically, any route ofadministration known in the art, e. g. buccal, sublingual, oral, rectal,transdermal, subcutaneous, intramuscular, intravenous, intraarterial,intraperitoneal, intrathecal, intralumbaginal or intradural, and anydosage regimen, e. g. as bolus or as continuous supply, may be employedto administer the agent.

The agent may be delivered simply as such or, preferably, in combinationwith any of a wide range of carriers and excipients, as known in theart, thereby forming a pharmaceutical composition. If desired, aconvenient drug targeting and/or delivery system may be used.Expediently, the agent and at least one carrier are combined into adosage form as known per se to those skilled in the art, e. g. into acontrolled or sustained release system. Basically, any carrier and/orexcipient compatible with the agent and any kind of dosage form may beused in the methods of the present invention. Suitable compounds andmethods are known in the art.

Thus, the present invention will be understood to also relate to themethods and uses relating to the manufacture of pharmaceuticalcompositions useful in the treatment of amyloidoses. In particular,amyloidoses according to the present invention comprise Alzheimer'sdisease and Down's syndrome.

In a particular embodiment of the invention, the treatment is arehabilitating and/or symptomatic treatment.

A “rehabilitating” treatment, as used herein, is, in particular, forproviding a benefit with regard to the patient's overall quality oflife.

As used herein, a “benefit” is any amelioration in relevant clinicalparameters or decrease in subjective suffering of the subject amenableto scoring that can be causally connected to a particular therapeuticmeasure. Expediently, the benefit is measured by comparing the relevantclinical parameters or the subjective suffering of the subject at a timepoint before treatment and at least one time point during or aftertreatment, and expressed in terms of a gain in quality-adjusted lifeyears or disability-adjusted life years (QALYs and DALYs).

The concept of “quality-adjusted life years” and “disability-adjustedlife years” is used extensively in the art to evaluate agents andmethods, in particular in the context of those diseases where morbidityand disability are medically and socially more of a concern thanmortality is, such as dementing diseases. Essentially, each year thelife time following treatment is multiplied with an index factor whichranges from 1.0 to indicate perfect quality of life, or zero disability,to 0.0 to indicate death, or complete disability, and the sum of theseproducts is compared to the value obtainable without treatment. Suitabledefinitions and methods for determining gains and losses in QALYs andDALYs, in particular with regard to dementing diseases such asamyloidoses, have been described in the art.

Thus, a benefit is preferably an increase in the aforementioned indexfactor. In a particular embodiment of the invention, the treatment ishence for providing a benefit to a subject suffering from anamyloidosis.

A “symptomatic” treatment, as used herein, is, in particular, atreatment directed to the abatement or relief of the symptoms of thedisease.

In a particular embodiment the present invention relates to a method forthe restoration of Aβ-impaired synaptic function and/or plasticity, inparticular long-term potentiation, in the subject.

In a further particular embodiment the present invention relates to amethod for the restoration of cognitive abilities, memory functionand/or performance of activities of daily life (ADL) capacity in thesubject.

As used herein, the terms “cognitive abilities”, “synaptic function”,“long-term potentiation” and “memory function” have the meanings as arewidely known and used in the art, and their quantificable values areconsidered as “normal” or “restored” when within the range which iscommonly to be expected, e. g. based on long-standing medical practice,appropriate clinical trials and/or biochemical analysis, for theindividual subject under consideration when compared to a representativepopulation of other subjects whose essential parameters otherwise agreewith those of said subject under consideration (peers of said subject).In particular, memory function is considered normal in a subject whensaid subject upon investigation by suitable means, e. g. short- and/orlong-time learning tests, shows no significant deficiencies with regardto memory in function in comparison to a control group matched inspecies, age, gender and optionally other factors acknowledged asrelevant to mental health, which are well-known to those skilled in theart, e. g. blood cholesterol levels, and/or psycho-social factors, e. g.educational and/or occupational background.

As used herein, the term “activities of daily living”, abbreviated“ADL”, is used to denote the essential manual and mental tasks andchores of everyday life, in particular those involving domains oflanguage (impairment thereof being known as “aphasia”), skilledmovements (impairment being known as “apraxia” and potentially leadingto total loss of control over the body in the final stages of thedisease), and the use of cognitive abilities such as recognition(impairment being known as “agnosia”, often accompanied bydisorientation and disinhibition, and sometimes also with behaviouralchanges) and higher-level intellectual functions (such asdecision-making and planning). These capacities can be assessed e. g.using questionnaire-based tests well-known in the art, such as theHodgkinson test (aka. “minimental state examination” or MMSE, comprisingthe recital of basic facts of everyday life) and the Folstein test (aka.“abbreviated mental test score” or AMTS, comprising, remembering thetime and place of the test, repeating lists of words, arithmetic,language use and comprehension, and copying a simple drawing) for basicmental functions and the John Hopkins Functioning Inventory (aka. JHFI)for basically motoric or movement-related abilities such as sitting,standing, walking, eating, washing, dressing etc.

The skilled person will be aware that in amyloidoses such as Alzheimer'sdisease the impairment of ADL capacity is dominated, in particular inits early and middle stages, by impairment of the intellectual ratherthan of motoric or sensory functions, and that even the latter, whenfound, is due to central rather than peripheral disturbances (e. g.“forgetting how to walk” rather than genuine organic paralysis).

According to another aspect, the present invention relates to a methodfor identifying an agent for the treatment of amyloidoses such asAlzheimer's disease, said method comprising determining whether acandidate compound exerts an agonistic effect on the P/Q typevoltage-gated presynaptic calcium channel, as disclosed above.

The invention will now be further illustrated by way of reference to thefollowing non-limiting examples and figures. Unless stated otherwise,the terms “A-Beta”, “Aβ₁₋₄₂”, “Aβ”, “aβ”, “glob” all denote the Aβ(1-42)globulomer described in reference example 2. “Kontrolle” means“control”.

DESCRIPTION OF THE FIGURES

FIG. 1: Effect of Aβ(1-42) globulomer on spontaneous synaptic activityas recorded from rat primary cultured hippocampal neurons by voltageclamp: (A) and (C) are recordings of spontaneously occurring synapticcurrents in a cultured hippocampal neuron (downward deflections indicatethe postsynaptic currents which are elicited by neurotransmitter releasefrom one or more presynaptic neurons; application of the globulomer andwashout (top trace) are indicated); (B) and (D) are the cumulativeprobability functions.

FIG. 2: Effect of Aβ(1-42) globulomer on the frequency of synapticcurrents.

FIG. 3: Effect of Aβ(1-42) globulomer on the frequency of mIPSCs in ofcells cultivated with 0.5 μM ω-conotoxin MVIIA to achieve synaptic P/Qpredominance (n=6): Number of synaptic events during 5 min relative tonon-Aβ globulomer treated cells. Left to right: (1) non-Aβ globulomertreated P/Q-dominated cells=reference, (2) P/Q-dominated cells treatedwith Aβ globulomer (at a concentration corresponding to approximately 1μM of Aβ monomer).

FIG. 4: Aβ(1-42) globulomer has no effect on the amplitude of mIPSCs ofcells cultivated with ω-conotoxin MVIIA to achieve synaptic P/Qpredominance: Average amplitude of synaptic events relative to non-Aβglobulomer treated cells. Left to right: (1) non-Aβ globulomer treatedP/Q-dominated cells=reference, (2) P/Q-dominated cells treated with Aβglobulomer (at a concentration corresponding to approximately 1 μM of Aβmonomer).

FIG. 5: Effect of ω-agatoxin on the frequency of mIPSCs in of cellscultivated with 0.5 μM ω-conotoxin MVIIA to achieve synaptic P/Qpredominance (n=3): Number of synaptic events during 5 min relative tonon-ω-agatoxin treated cells. Left to right: (1) non-ω-agatoxin treatedP/Q-dominated cells=reference, (2) P/Q-dominated cells treated with 0.5μM ω-agatoxin.

FIG. 6: No additive effect on the frequency of mIPSCs in of cellscultivated with 0.5 μM ω-conotoxin MVIIA to achieve synaptic P/Qpredominance after blockade of P/Q-channels by ω-agatoxin (n=6): Numberof synaptic events during 5 min relative to non-Aβ globulomer treatedcells. Left to right: (1) non-Aβ globulomer treated P/Q-dominated cells(ω-agatoxin only)=reference, (2) P/Q-dominated cells treated with Aβglobulomer (at a concentration corresponding to approximately 1 μM of Aβmonomer) after pre-treatment with 0.5 μM ω-agatoxin.

FIG. 7: No effect of globulomer on the amplitude of mIPSCs when P/Qchannels of P/Q-dominated cells are already blocked by 0.5 μM ω-agatoxinIVA (n=6): Number of synaptic events during 5 min relative to non-Aβglobulomer treated cells. Left to right: (1) non-AP globulomer treatedP/Q-dominated cells (ω-agatoxin only)=reference, (2) P/Q-dominated cellstreated with Aβ globulomer (at a concentration corresponding toapproximately 1 μM of Aβ monomer) after pre-treatment with 0.5 μMω-agatoxin.

FIG. 8: Agatoxin does not impair spontaneous synaptic activity incultures that lack functional P/Q-type Ca⁺⁺ channels: Number of synapticevents during 5 min was set to 100% for each cell analysed. The rightbar indicates the relative number of synaptic events in each cell afterapplication of 0.5 μM ω-agatoxin.

FIG. 9: Globulomer does not impair spontaneous synaptic activity incultures that lack functional P/Q-type Ca⁺⁺ channels: Number of synapticevents during 5 min relative to non-Aβ globulomer treated cells was setto 100% for each cell analysed. The right bar indicates the relativenumber of synaptic events in each cell after application of Aβglobulomer (at a concentration corresponding to approximately 1 μM of Aβmonomer).

FIG. 10: Suppression of spontaneous synaptic currents by Aβ(1-42)globulomer and its reversal by the P/Q channel agonist roscovitine:Number of synaptic events during 5 min relative to non-Aβ globulomertreated P/Q-dominated cells. Left to right: (1) non-Aβ globulomertreated P/Q-dominated same cells=reference, (2) P/Q-dominated same cellstreated with Aβ globulomer (at a concentration corresponding toapproximately 1 μM of Aβ monomer), (3) P/Q-dominated same cells treatedsimultaneously with Aβ globulomer (at a concentration corresponding toapproximately 1 μM of Aβ monomer) and 20 μM roscovitine.

FIG. 11: No effect on the amplitude of spontaneous synaptic currents ofthe P/Q channel agonist roscovitine: Average amplitude of synapticevents relative to non-Aβ globulomer treated P/Q-dominated cells. Leftto right: (1) non-Aβ globulomer treated P/Q-dominated samecells=reference, (2) P/Q-dominated same cells treated with Aβ globulomer(at a concentration corresponding to approximately 1 μM of Aβ monomer),(3) P/Q-dominated same cells treated simultaneously with Aβ globulomer(at a concentration corresponding to approximately 1 μM of Aβ monomer)and 20 μM roscovitine.

FIG. 12: The effect of Aβ(1-42) globulomer on spontaneous synapticactivity of P/Q-dominated cells can be reversed by the P/Q channelagonist roscovitine: Synaptic potentials over time. Arrows indicate thetime points when Aβ globulomer (at a concentration corresponding toapproximately 1 μM of Aβ monomer) and 20 μM roscovitine, respectively,were added.

FIG. 13: Reducing effect of Aβ globulomer on the amplitude ofpharmacologically isolated P/Q-type calcium channels: Traces representmembrane currents after activation of P/Q-type channels by adepolarizing voltage step. Left to right: (1) P/Q-current under controlconditions, (2) P/Q-current of the same cell after application of Aβglobulomer (at a concentration corresponding to approximately 1 μM of Aβmonomer), (3) P/Q-current of the same cell after washout of Aβglobulomer.

FIG. 14: Effect of Aβ(1-42) globulomer on the pharmacologically isolatedP/Q current at different time points: Average amplitude of P/Q-mediatedcurrent amplitude relative to non-Aβ globulomer treated P/Q-dominatedcells. Left to right: (1) non-Aβ globulomer treated samecells=reference, (2) same cells 10 min after treatment with Aβglobulomer (at a concentration corresponding to approximately 1 μM of Aβmonomer), (3) same cells 15 min after treatment with Aβ globulomer (at aconcentration corresponding to approximately 1 μM of Aβ monomer).

FIG. 15: Effect of 0.5 μM ω-agatoxin IVA on the pharmacologicallyisolated P/Q current at different time points: Average amplitude of P/Qcurrents relative to non-ω-agatoxin treated P/Q-dominated same cells.Left to right: (1) non-ω-agatoxin treated P/Q-dominated same cells=reference, (2) P/Q-dominated cells 10 min after treatment with 0.5 μMω-agatoxin, (3) P/Q-dominated cells 15 min after treatment with 0.5 μMω-agatoxin.

FIG. 16: Effect of Aβ on the pharmacologically isolated P/Q current atdifferent time points, revealing the effect of washing out the Aβglobulomer: Average amplitude of P/Q-mediated current relative to non-Aβglobulomer treated P/Q-dominated cells. Left to right: (1) non-Aβglobulomer treated P/Q-dominated cells=reference, (2) P/Q-dominatedcells 10 min after treatment with Aβ globulomer (at a concentrationcorresponding to approximately 1 μM of Aβ monomer), (3) P/Q-dominatedcells 15 min after treatment with Aβ globulomer (at a concentrationcorresponding to approximately 1 μM of Aβ monomer), (4) P/Q-dominatedcells treated with Aβ globulomer (at a concentration corresponding toapproximately 1 μM of Aβ monomer) after washing out the Aβ globulomer.

FIG. 17: Effect of Aβ on spontaneous synaptic activity in thehippocampal slice: Number of synaptic events during 5 min relative tonon-Aβ globulomer treated tissue. Left to right: (1) non-Aβ globulomertreated same slice=reference, (2) same slice treated with Aβ globulomer(at a concentration corresponding to approximately 1 μM of Aβ monomer).

FIG. 18: Spontaneous synaptic activity is reversibly suppressed byAβ(1-42) globulomer. Original recording of spontaneously occurringsynaptic currents in a cultured hippocampal neuron before (top), during(middle) and after (bottom) application of Aβ(1-42) globulomer.

FIG. 19: Effects of Aβ(1-42) globulomer on different types of synapticcurrents in cultured hippocampal neurons. White bars: effect of Aβ(1-42)globulomer; black bars: washout for at least 10 min. A: Reduction ofevent frequency as percentage of previously recorded control currents(1.0). B: Effects of Aβ(1-42) globulomer on median amplitude of therespective currents. sPSCs: spontaneously occurring pharmacologicallynaive postsynaptic currents; mPSCs: pharmacologically naive miniaturepostsynaptic currents recorded in the presence of TTX; mIPSCs: miniatureinhibitory postsynaptic currents; mEPSCs: spontaneously occurringexcitatory postsynaptic currents; mEPSCs: miniature excitatorypostsynaptic currents.

FIG. 20: Stability of GABA_(A) receptor-mediated currents towardsAβ(1-42) globulomer. A: Repetitive application of 100 μM GABA to acultured hippocampal neuron yields stable inward current before, during,and after application of the oligomer. B: Enlarged view of currenttraces marked with * in A. Note the stability of response in the absence(left) and presence (right) of Aβ(1-42) globulomer. C: Time course ofGABA-induced currents from 5 cells recorded in control solution (dashedline) and from 3 neurons where Aβ(1-42) globulomer was applied(continuous line, time of application indicated by bar). Amplitudesnormalized to the last GABA-induced current before application ofAβ(1-42) globulomer.

FIG. 21 Suppression of P/Q-type calcium currents by Aβ(1-42) globulomer.A: Time course of current amplitudes upon application of globulomer.Currents were elicited by voltage steps to −10 mV. B: Example traces ofP/Q-type currents before, during and after globulomer.

FIG. 22 Steady-state activation and inactivation parameters of P/Qcurrents. A: Current/voltage relationship before globulomer (squares)and during Aβ₁₋₄₂ (triangles). A reduction of the current amplitudesover the entire voltage-range, were the current could be activated, wasobserved following application of the globulomer. B & C: No differencein steady-state activation (B) and inactivation curves (C) for P/Qchannel-mediated barium currents in the absence and presence of Aβ(1-42)globulomer. D: A significant decrease in maximal conductance (g_(max))of the P/Q channels was induced by Aβ(1-42) globulomer.

FIG. 23 Pharmacological modulation of the effect of Aβ(1-42) globulomerby agents interacting with P/Q-type calcium channels. A: Effects ofAβ(1-42) globulomer on frequency of mixed synaptic currents. B: Effectson median amplitude. Values are given relative to data in controlsolution. Note suppression of the effect by ω-agatoxin and partialrecovery of event frequency by roscovitine.

FIG. 24 Pharmacological modulation of the effect of Aβ(1-42) globulomerby agents interacting with P/Q-type calcium channels. Left to right: (1)frequency of mixed synaptic currents of non-Aβ(1-42) globulomer treatedcultivated hippocampal cells, (2) frequency of mixed synaptic currentsafter application of Aβ(1-42) globulomer (at a concentrationcorresponding to approximately 1 μM of Aβ monomer), (3) frequency ofmixed synaptic currents after application of Aβ(1-42) globulomer (at aconcentration corresponding to approximately 1 μM of Aβ monomer) and 15μM isoproterenol.

FIG. 25 Enhancing P/Q calcium currents by roscovitine prevents/reverseschronic Aβ globulomer-induced deficits on evoked synaptic transmissionin hippocampal tissue (slice cultures). Recordings were performed afterincubation with Aβ(1-42) globulomer (at a concentration corresponding toapproximately 1 μM of Aβ monomer), Aβ(1-42) globulomer (at aconcentration corresponding to approximately 1 μM of Aβ monomer)+20 μMroscovitine, or control (SDS).

FIG. 26 Enhancing P/Q calcium currents by a roscovitine analogueprevents/reverses chronic Aβ globulomer-induced deficits on evokedsynaptic transmission in hippocampal tissue (slice cultures). Recordingswere performed after incubation with Aβ(1-42) globulomer (at aconcentration corresponding to approximately 1 μM of Aβ monomer),Aβ(1-42) globulomer (at a concentration corresponding to approximately 1μM of Aβ monomer)+20 μM roscovitine analogue A, or control (SDS).

FIG. 27 Effect of extracellular Ca²⁺ on sPSC frequency after treatmentwith Aβ(1-42) globulomer: Original recording of sPSCs before (control in1 mM Ca²⁺), after addition of Aβ(1-42) globulomer (glob in 1 mM Ca²⁺)and after subsequent elevation of Ca²⁺-concentration (glob in 4 mMCa²⁺). B: Reduction of event frequency after application of Aβ(1-42)globulomer (p<0.05; n=6) and partial recovery after elevation of Ca²⁺from 1 mM to 4 mM. Values are given as percentage of control currents.C: Event frequency of single cells (n=6) after application of Aβ(1-42)globulomer and after subsequent elevation of Ca²⁺ from 1 mM to 4 mM.Values are given as percentage of control currents. D: No difference inmedian amplitude after application of Aβ(1-42) globulomer (n=6) andafter subsequent elevation of Ca²⁺. Values are given as percentage ofcontrol currents. E: Original recordings of massive discharges directlyafter Ca²⁺ elevation for the cell shown in A. These currents wererejected from analysis.

FIRST SERIES OF EXPERIMENTS REFERENCE EXAMPLE 1 Determination ofSynaptic Potentials

Neuronal cells from the rat hippocampus were obtained and cultured inaccordance with methods known per se in the art (Banker G A, Cowan W M,Brain Res. 1977 May 13; 126(3):397-42). Cultured neurons showspontaneous postsynaptic currents (PSCs), i. e. spontaneous PSCs and, inthe presence of the sodium channel blocker tetrodotoxin, miniature PSCs.As mentioned above, the influx of Ca⁺⁺ through presynaptic ion channelssuch as the N, P/Q and R type voltage-gated presynaptic calcium channelsis what causes the release of neurotransmitter from preformed vesiclesin presynaptic terminals. The measured signal reflects the currentresponse of the postsynaptic cell to the release of such transmitters,e.g. gamma-aminobutyric acid or glutamate.

For measurements, primary cell cultures were transferred to a recordingchamber mounted on a microscope (Olympus CKX1) and were immersed at roomtemperature into a buffered solution consisting of 156 mM NaCl, 2 mMKCl, 2 mM CaCl₂, 1 mM MgCl₂, 16.5 mM glucose and 10 mM HEPES at a pH of7.3. The osmolarity of the solution was 330 mosmol.

Electrodes were produced by pulling from borosilicate capillaries(available from Science Products) with a horizontal pipette pullingdevice (P-97 from Sutter Instruments). After filling with theintracellular solution, the final resistance of the electrodes was from2 to 5 MΩ. The intracellular solution consisted of either (forrecordings of miniature PSCs) 100 mM KCl, 10 mM NaCl, 0.25 mM CaCl₂, 5mM EGTA, 40 mM glucose, 4 mM MgATP and 0.1 mM NaGTP at a pH of 7.3, or(for recording of calcium currents) 110 mM CsCl, 10 mM EGTA, 25 mMHEPES, 10 mM tris-phosphocreatine, 20 U/ml creatine phosphokinase, 4 mMMgATP and 0.3 mM NaGTP.

All test compounds were applied either by bath perfusion or by additionto the bath by means of a micropump connected to a manually guidedpipette.

All recordings of miniature PSCs were made in the presence of 0.5 μMtetrodotoxin (TTX; available from Tocris Bioscience) to block the Na⁺and K⁺ channels in the neuronal cell membrane which would otherwise alsoinfluence the electrical status of the membrane. For calcium currentrecordings the extracellular solution contained 140 mM TEA-CI (to blockK⁺-channels) 10 mM BaCl₂, 0.5 μM TTX, 10 mM HEPES and 20 mM glucose at apH 7.3. When required, ω-conotoxin MVIIA (available from Alomone Labs,Jerusalem, Israel) was added to a final concentration of 0.5 μM to blockN type voltage-gated presynaptic Ca⁺⁺ channels, thereby“pharmacologically isolating” the ion fluxes through the P/Q typevoltage-gated presynaptic calcium channel. If necessary, L-typevoltage-gated calcium channels were blocked by addition of 10 μMnifedipine.

To mimic the effect of Aβ globulomer as P/Q type blocker, ω-agatoxin IVA(available from Alomone Labs, Jerusalem, Israel) was added to a finalconcentration of 0.5 μM to specifically block the P/Q type voltage-gatedpresynaptic Ca⁺⁺ channels of the sample cell.

All substances were stored as lyophilized powders at −20 ° C. Stocksolutions were prepared with vehicles appropriate for the solubility (i.e. immersion solution). Vehicle was destilled water or standardextracellular solution for all drugs except nifedipine, which wasdissolved in ethanol, and roscovitine, which was dissolved in dimethylsulfoxide (DMSO). The final concentration of the solvents in theAβ-globulomer solvent buffer which was applied to neurons was <1%0 andthe final concentration of DMSO was <1.5%o.

Whole-cell patch-clamp recordings (sPSCs and mPSCs) were conducted in amanner essentially known per se (see, e.g., Sakmann B and Neher E.Single-Channel Recording. Springer US, 97 A.D.) at a holding potentialof −70 mV using an EPC7 amplifier (available from HEKA Electronics).Signals were filtered at 3 kHz and sampled at 20 kHz.

After formation of a seal, rupture of the membrane by the electrode andestablishment of the whole-cell configuration, the perfusion of the bathwas stopped, and the substances to be tested were injected into the bathusing a custom-made syringe pump.

The sPSCs or mPSCs were then recorded for 10 minutes giving the controlvalues before any toxins were added.

For the selective determination of P/Q type voltage-gated presynapticcalcium channel currents, the cells were activated in a manner known perse (see Yan et al., 2002, supra) by a voltage protocol, where the cellswere excited by depolarization to −10 mV for 50 ms every 20 sec. Afterthe formation of the whole-cell configuration, currents increasedsteadily until they had reached a stable amplitude level. After thisstable amplitude level had been established, the effects of differenttest compounds on the rate of ion flux were observed and expressed interms of the normalized mean P/Q amplitude and standard error of themean SEM. Frequency and amplitude of synaptic currents were calculatedoffline using a template-based algorithm (custom made routine within theSignal and Spike software, purchased from CED Inc., Cambridge, UK).

When desired, the measurement was evaluated at several timepoints andoptionally after a washout. Student's t-test was applied to determinesignificance, p<0.05 being considered as indicative of significantdifferences.

REFERENCE EXAMPLE 2 Generation of Aβ Globulomer

An Aβ(1-42) globulomer preparation with an apparent molecular weight of38/48 kDa as determined by SDS-PAGE was obtained as described in Example6b of WO2004/067561. Essentially, Aβ monomer was pretreated with HFIPfor dissolving hydrogen bonds, then diluted and further incubated in thepresence of 0.2% SDS, followed by isolation of the thus formedglobulomer.

EXAMPLE 3 Inhibitory Effect of Aβ Globulomer on Spontaneous SynapticActivity

Using acute application of the P/Q channel blocker ω-agatoxin as anegative control and cells untreated with regard to the P/Q typevoltage-gated presynaptic calcium channel as a positive control, theeffects of Aβ(1-42) globulomer on the frequency of spontaneous synapticevents in cultured hippocampal neurons treated with ω-conotoxin toachieve synaptic dominance of the P/Q type channel, as described inReference Example 1, were observed.

Aβ globulomer, obtained as described in Reference Example 2, was testedaccording to the procedure described in Reference Example 1 for channelfunction inhibitors such as ω-agatoxin. In the presence of ω-agatoxin,Aβ globulomer had no further effect on synaptic activity, indicatingthat the effects of both agents involved a common mechanism. A total of200 μl Aβ-globulomer solvent buffer comprising a Aβ(1-42) globulomerconcentration corresponding to approximately 2 μM of Aβ monomer wasadded to the bath (previous volume 200 μl), resulting in a finalAβ(1-42) globulomer concentration corresponding to approximately 1 μM ofAβ monomer. Based on the assumption that the Aβ(1-42) globulomerconsists of 12 Aβ(1-42) monomers a final Aβ(1-42) globulomerconcentration of approximately 83 nM can be calculated. Measurements ofsynaptic activity were then taken.

Results are shown in FIGS. 1-7, demonstrating that the Aβ globulomerinhibits the frequency of spontaneous synaptic events with an efficiencyapproaching that of the strong P/Q inhibitor ω-agatoxin but has no orlittle effect on the amplitude of the synaptic events. Thus, Aβ(1-42)globulomer reduces synaptic activity, most likely by a presynapticmechanism, which shares crucial elements with the effect of ω-agatoxin.

These results were verified by subjecting the Aβ(1-42) globulomercontaining Aβ-globulomer solvent buffer to ultrafiltration with a filterhaving a molecular cutoff size of 5 kDa for globular proteins. Theresulting solvent buffer contained no detectable amounts of Aβglobulomer protein prior to bringing it into contact with the cells. Theultrafiltrate had no effect on the synaptic events (see FIG. 2),indicating that the agent responsible for reducing the frequency ofspontaneous synaptic events was unable to pass ultrafilters.

Furthermore, the effect of Aβ(1-42) globulomer is absent in cellspredominantly expressing presynaptic N-type calcium channels. Resultsare shown in FIGS. 8 and 9, demonstrating that in the N-dominated cellsno reduction of the frequency nor any reduction in amplitude is achievedby either ω-agatoxin or Aβ globulomer, i. e. that both ω-agatoxin and Aβglobulomer target the P/Q type voltage-gated presynaptic calciumchannel.

EXAMPLE 4 Rescue of Spontaneous Synaptic Activity by Roscovitine

Using the Aβ(1-42) globulomer of Reference Example 2 as a negativecontrol and cells untreated with regard to the P/Q type voltage-gatedpresynaptic calcium channel as a positive control, the effects of theP/Q type voltage-gated presynaptic calcium channel activator roscovitineon the Aβ globulomer-induced reduction of the frequency of spontaneoussynaptic events in cultured hippocampal neurons treated withω-conotoxin, as described in Reference Example 1, were observed.

Roscovitine was used at a final concentration of 20 μM, by adding itsimultaneously with Aβ(1-42) globulomer (final concentration of Aβglobulomer corresponding to approximately 1 μM of Aβ monomer).Roscovitine is known (Zhen Yan et al., J. Physiol. 540: 761-770 (2002))to slow down the inactivation of the P/Q type voltage-gated presynapticcalcium channel, i. e. to extend the time for which a channel, onceopened, remains in the open state, thereby increasing the calcium ionflow through the P/Q type voltage-gated presynaptic calcium channel.

Results are shown in FIGS. 10 and 11, demonstrating that a P/Q typevoltage-gated presynaptic calcium channel activator is capable ofrestoring the frequency of spontaneous synaptic events under theinfluence of Aβ globulomer to almost that of untreated cells, i. e.,that a P/Q activator may be used to reverse the detrimental effects ofAβ globulomer.

REFERENCE EXAMPLE 5 Direct Determination of the Activity of the P/Q TypeVoltage-Gated Presynaptic Calcium Channel, and of Inhibitory andActivating Influences, by the Voltage-Clamp Method

Cells were prepared and subjected to measurement of membrane currents bythe voltage-clamp method basically as described in Reference Example 1,the difference being essentially that all irrelevant (non-P/Q type) ionchannels of the cells were blocked chemically, thereby allowing fordirect determination of the ion fluxes rather than of the resultingIPSCs. Blocking of the irrelevant channels was achieved using thefollowing additions to the bath or electrode solution:

Compound Concentration Channel blocked TEA-Cl 140 mM I[K⁺] BaCl₂ 10 mMCsCl (in the pipette) 110 mM Nifedipine 10 mM L-type Ca⁺⁺ channelω-conotoxin MVIIA 0.5 mM N-type Ca⁺⁺ channel Tetrodotoxin 0.5 Na⁺channels

The Ba⁺⁺ also served as the charge carrier (i. e. substrate replacement)for the P/Q type voltage-gated presynaptic Ca⁺⁺ channel, with theadditional advantage that conductance through this channel and hence thesensitivity of the assay were thereby increased to approximatelytenfold. This made it possible to directly detect ion fluxes throughP/Q-channels in somatic recordings.

In order to prevent the “run down” of Ca⁺⁺ currents in the samples, theelectrode solution also comprised, in addition to the substances listedabove, 10 mM tris-phosphocreatinine and 20 U/ml creatine phosphokinase,which together served as an ATP regenerating system preventing“run-down”, i.e. decline due to a gradual loss of channel conductance,of the observed currents. ATP is needed to maintain the conductance ofthe calcium channels over time intervals longer than several minutes,allowing to conduct the described pharmacological experiments withsufficiently stable calcium currents.

EXAMPLE 6 Direct Effect of Aβ Globulomer on the P/Q Type Voltage-GatedPresynaptic Calcium Channel in Cultured Cells

Using ω-agatoxin as a negative control and cells untreated with regardto the P/Q type voltage-gated presynaptic calcium channel as a positivecontrol, the effects of Aβ(1-42) globulomer of Reference Example 2 (at aconcentration corresponding to approximately 1 μM of Aβ(1-42) monomers)on calcium flux in hippocampal neurons treated with ω-conotoxin weredirectly observed as described in Reference Example 5.

Recordings were taken at 10 min and 15 min and optionally after awashout. Typical results are shown in FIGS. 13-16. These findingsdemonstrate that Aβ globulomer directly inhibits the activity of the P/Qtype voltage-gated presynaptic calcium channel and cannot be readilywashed out after binding to the P/Q type voltage-gated presynapticcalcium channel. They further demonstrate that Aβ globulomer impedes, bydecreasing the amplitude of the calcium flux, the initiation of synapticcurrents.

EXAMPLE 7 Direct Effect of Aβ Globulomer on the P/Q Type Voltage-GatedPresynaptic Calcium Channel In Situ

To verify whether the effect of the globulomer on neurons in cellcultures also takes place in the more organotypic slice-preparation ofthe hippocampus, synaptic currents were determined in this tissue.

400 μm thick slices were prepared from freshly dissected hippocampi ofthe mouse using a method known per se (Dingledine R. Brain Slices. NewYork: Plenum Press, 1983). CA1 pyramidal cells were patched andspontaneous synaptical currents were recorded prior and afterapplication of Aβ(1-42) globulomer via an Eppendorff pipette.

Typical results are shown in FIG. 17. These findings demonstrate thatthe mechanism for Aβ globulomer mediated inhibition disclosed herein isalso valid in situ.

SECOND SERIES OF EXPERIMENTS REFERENCE EXAMPLE 8 Cell Culture

Primary hippocampal cell cultures were prepared from Wistar rat embryosat the embryonic age E19 in accordance with the protocol describedearlier by Banker and Cowan (1977). Briefly, pregnant rats were deeplyanesthetized by ether narcosis and decapitated. Embryos were rapidlyremoved and brains were dissected under constant cooling with chilled(˜4° C.) phosphate buffered saline (PBS). Then both hippocampi weretaken out and washed twice with ice-cold PBS followed by a wash with PBSat room temperature. Hippocampi were triturated using three siliconizedpipettes with decreasing tip diameters. Cells were then plated oncoverslips (density 60000 cells/coverslip) coated with 0.01%poly-L-lysine solution and stored at 37° C. in an incubator gassed with5% CO₂ in normal air. The culture medium contained 0.25%penicilline/streptomycine, 2% B27, 0.25% L-glutamine (Gibco, Karlsruhe,Germany). Throughout culturing, we added 0.5 μM/L ω-conotoxin MVIIA tothe culture medium to block N-type calcium channels and to stabilize theexpression of P/Q-type currents. Cells were cultured for 14 to 28 daysuntil used for experiments.

REFERENCE EXAMPLE 9 Current Recording

Currents were measured under whole-cell voltage-clamp conditions at roomtemperature using borosilicate pipettes of 2-4 MΩ resistance. Electrodesolution contained (in mM/l): NaCl 10, KCl 100, CaCl₂ 0.25, EGTA 5,HEPES 10, glucose 40 (pH set at 7.3) when used for recordings ofsynaptic events. A low-chloride solution was used for experiments inwhich GABA induced currents were elicited, which consists of (mM):Cs-gluconate 130, CsCl 10, CaCl₂ 0.5, MgCl₂ 2, EGTA 10, HEPES 10, Mg-ATP2 (pH: 7.3). Using this solution the calculated equilibrium potentialfor chloride-ions was −54 mV. During calcium current measurements thesolution contained in (mM): CsCl 110, EGTA 10, HEPES 25,tris-phosphocreatine 10, Mg-ATP 4, Na-GTP 0.3 and 20 units/mlcreatine-phosphokinase at pH 7.3. Osmolarity was adjusted to 295mosmol/l, when necessary, by adding glucose. Bath solutions contained(in mM): NaCl 156, KCl 2, CaCl₂ 2, MgCl₂, Glucose 16.5, HEPES 10 (pH setto 7.3) for recordings of synaptic events and TEA-Cl 140, BaCl₂, 10,HEPES 10, and Glucose 20 at a pH: 7.3 for calcium currents,respectively. The bath perfusion was stopped for 10 min prior to theapplication of the Aβ(1-42) globulomer and baseline activity wasrecorded. Subsequently, Aβ(1-42) globulomer (164 nM in respect to the12mer complex) was added to the bath by means of a micro pump, yieldinga final concentration of 82 nM. TTX, ω-agatoxin IVA, ω-conotoxin MVIIA,roscovitine (Alomone Labs, Jerusalem, Israel), and nifedipine (Sigma,Deisenhofen, Germany) were added directly to the bath solution at theconcentrations indicated.

Currents were measured with an Axopatch 200B (Axon Instruments, UnionCity, US) or an EPC-7 amplifier (HEKA, Lambrecht, Germany), digitized bya CED 1401 micro analog/digital converter (CED, Cambridge, UK) andstored on a PC (sample frequency 20 kHz). All recorded currents werelow-pass filtered with a cut-off frequency of 3 kHz. Capacitivetransients and series resistances were compensated on-line (˜50-60%compensation) during the calcium current measurements. No compensationwas performed during recordings of synaptic events. Data were evaluatedoff-line using Spike5 and Signal3 software (CED, Cambridge, UK). Allcalcium current traces were corrected for aspecific linear leak(reversal potential 0 mV) determined at holding potential using ±5 mVpotential steps.

REFERENCE EXAMPLE 10 Current Analysis

All cells were voltage clamped at a holding potential of −80 mV, andcalcium ions were substituted by Barium ions to increase the amplitudeof the current flow through the calcium channels. Peak amplitudes of thecurrents (I) evoked with the activation protocol were plotted as afunction of membrane potential (V). The resulting IV-relations werefitted with a combination of a first order Boltzmann activation functionand the Goldman-Hodgkin-Katz (GHK) current-voltage relation (Kortekaasand Wadman, 1997):

$\begin{matrix}{{{I(V)} = {V\frac{g_{\max}}{1 + {\exp \left( \frac{V_{h} - V}{V_{c}} \right)}}\frac{{\left\lbrack {Ba}^{+} \right\rbrack_{in}/\left\lbrack {Ba}^{+} \right\rbrack_{out}} - {\exp \left( {{- \alpha}\; V} \right)}}{1 - {\exp \left( {{- \alpha}\; V} \right)}}}}{{{with}\mspace{14mu} a} = {{{F/{RT}}\mspace{14mu} {and}\mspace{14mu} g_{\max}} = {\alpha \; {{FP}_{0}\left\lbrack {Ba}^{+} \right\rbrack}_{{out},}}}}} & \lbrack 1\rbrack\end{matrix}$

where g_(max) is the maximal membrane conductance (which is proportionalto the maximal permeability and the extracellular concentration ofbarium), V_(h) is the potential of half maximal activation and V_(c) isproportional to the slope of the curve at V_(h). F represents theFaraday constant, R the gas constant, P₀ is the maximal permeability,and T the absolute temperature. The intracellular concentration of Ba²⁺was assumed to be 0.01 μM. Assuming higher values of up to 0.1 mM didnot significantly change the resulting values of the parameters.

The voltage dependence of steady state inactivation of the bariumcurrent was estimated from the relation of peak current amplitude versusthe pre-potential. This relation was well described by a Boltzmannfunction, which normalized the current:

$\begin{matrix}{{N(V)} = {{\frac{I(V)}{I_{\max}}\mspace{14mu} {where}\mspace{14mu} {I(V)}} = \frac{I_{\max}}{1 + {\exp \left( \frac{V_{h} - V}{V_{c}} \right)}}}} & \lbrack 2\rbrack\end{matrix}$

where N(V) is the level of steady state inactivation determined from thecurrent amplitude I(V) normalized to I_(max), V is the pre-pulsepotential, V_(h) is the potential of half maximal inactivation and V_(c)is a factor proportional to the slope of the curve at V_(h).

REFERENCE EXAMPLE 11 Synaptic Events

For these recordings, all cells were voltage clamped at a holdingpotential of −70 mV. Synaptic events triggered by the release of GABAwere inwardly directed (E_(Cl)˜−10 mV) due to the use of high chlorideconcentrations in the pipette and the bath. Routinely, 10 min ofbaseline activity was acquired, serving as control data, before any drugapplication was started. Synaptic events were then analyzed off-line forfrequency and amplitude, using a custom-made, template based algorithm.

REFERENCE EXAMPLE 12 Statistics

Values are presented as the mean±standard error of the mean (SEM).Statistical comparisons were made with Student's t-test. A p-value<0.05was used to indicate significant differences.

EXAMPLE 13 Aβ(1-42) Globulomer Reduces Spontaneous Synaptic Activity inHippocampal Cell Cultures

Spontaneous synaptic was measured activity in cultured hippocampalneurons using whole-cell voltage clamp techniques (V_(hold)=−70 mV).Under our ionic conditions, all synaptic events appeared as inwardcurrents (spontaneous postsynaptic currents; sPSCs) with a meanfrequency of 189±63/min (n=13). Bath-application of 82 nM Aβ(1-42)globulomer (globulomer molarities calculated with respect to the 12mercomplex) rapidly reduced the frequency of sPSCs to 38±5% of control(p<0.05; n=13; FIG. 18). This effect was partially reversible uponwashout in 2 of 3 cells tested (61±16%). The median amplitude of eventswas 310±168 pA and was reduced to 84±10% under Aβ(1-42) globulomer(p<0.05; n=14; FIG. 19). Similar—but slightly weaker—effects were seenafter application of 8.2 nM Aβ(1-42) globulomer (frequency reduced to63±9%; p<0.05; median amplitude 94±5% of control, n=8, n.s.). Thus, thesuppression of spontaneous synaptic activity by Aβ(1-42) globulomer isdose-dependent and starts at low nanomolar concentrations. Inputresistance was not affected by Aβ(1-42) globulomer (control: 120.9±13.6MO; Aβ(1-42): 131.6±13.7 MΩ).

Suppression of synaptic currents by an agent may be caused by changes inneuronal activity or, alternatively, by specific synaptic interactions.It was therefore tested for effects of Aβ(1-42) globulomer on activedischarge properties by recording action potentials in current clampmode. Action potentials elicited by current injection showed nodifference in amplitude, shape or kinetics when compared before andafter Aβ(1-42) globulomer application. In detail, the threshold forfiring was −22.5±8.2 mV vs. −24.2±9.8 mV, and the amplitude of the AP(baseline to peak) amounted to 119.9±11.2 vs. 110.9±16.7 mV. Likewise,kinetic parameters did not differ: values for the half-width time were3.5±1.6 ms vs. 4.0±2.9 ms, maximal rate of rise 100.5±46.4 V/s vs.84.2±50.0 V/s and maximal rate of repolarization 46.0±18.6 V/s vs.47.4±19.3 V/s (n=16 action potentials from 4 cells before and afterAβ(1-42) globulomer respectively. It thus appears that the alteration ofsynaptic activity by Aβ(1-42) globulomer may be caused by a directinteraction with pre- or postsynaptic proteins, rather than by anunspecific alteration of cellular excitability.

This hypothesis was corroborated by recordings of spontaneouslyoccurring miniature post-synaptic currents (mPSCs) in the presence ofTTX. Similar to spontaneous “macroscopic” PSCs, these currents werereduced in frequency by 82 nM Aβ(1-42) globulomer (yielding 56±9% ofcontrol; p<0.05; FIG. 19). However, the amplitude of mPSCs was unaltered(median amplitude 31.1±4.0 pA under control conditions vs. 30.2±5.2 pAin the presence of Aβ(1-42) globulomer, n=6). Upon washout for 10minutes, the effect on event frequency recovered partially to 77±7.6% ofcontrol, n=6, wash: 4/6). Together, these data suggest that Aβ(1-42)globulomer interferes with the presynaptic machinery of transmitterrelease.

EXAMPLE 14 Effects on Spontaneous and Miniature Inhibitory PostsynapticCurrents

Pharmacologically naive synaptic currents reflect a mixture ofglutamatergic (excitatory) and GABAergic (inhibitory) events. In orderto differentiate between these components, inhibitory postsynapticcurrents were isolated by adding CNQX (20 μM) and DL-APV (30 μM) to thebath solution. The frequency of spontaneously occurring IPSCs wassuppressed by Aβ(1-42) globulomer (yielding 64±5% of control; p<0.05;n=12) and the median amplitude was reduced to 82±2% of control (p<0.05).These reductions could be reversed to some degree following withdrawalof the globulomer (frequency: 90±12%; amplitude: 94±2%). Miniatureinhibitory postsynaptic currents (mIPSCs, recorded in 0.5 μM TTX) didalso show a similar reduction of frequency after application of Aβ(1-42)globulomer (52±10% of control; p<0.05; n=6). This effect was partiallyreversible upon washout, yielding 68±12% of control (FIG. 19). Inaddition, a reduction of mIPSC amplitude was observed (81±6% of control;p<0.05; no washout in 3/3 cells (85±6%)).

EXAMPLE 15 Effects on Postsynaptic GABA_(A) Receptors

In order to test for potential effects of Aβ(1-42) globulomer onpostsynaptic GABA_(A) receptors, a high (100 μM) concentration of GABAwas applied by brief pressure-pulses directly onto the cell. Repetitiveapplication of GABA to cultured cells elicited large (>1 nA) inwardcurrents which showed only minor rundown with time. This behaviour wasunaltered after application of Aβ(1-42) globulomer for 5 min, indicatingthat GABA_(A) receptors are not affected by the agent (FIG. 20).

EXAMPLE 16 Effects on Spontaneous and Miniature Excitatory PostsynapticCurrents

Finally, excitatory synaptic currents (EPSCs) were isolated in thepresence of 5 μM gabazine (a GABA_(A) receptor antagonist). Basalfrequency of these events was 386±124/min. Their frequency was reducedby Aβ(1-42) globulomer to 14±4% of control (p<0.05; n=6; FIG. 19).Likewise, the amplitude was reduced to 79±4% of control (n=6; p<0.05;FIG. 19). The effect was partially reversible during washout (frequencyincreasing to 52±19% of control, amplitude to 96±6%; n=6). The frequencyof miniature EPSCs was likewise suppressed to 57±9% of control (n=6;p<0.05), while the amplitude of mEPSCs remained stable (96±3% ofcontrol). The frequency suppression did not recover upon wash-out(54±8%; n=6). Together, these data indicate that Aβ(1-42) globulomerdepresses vesicular release at GABAergic and glutamatergic synapses,most likely by decreasing the probability of vesicle exocytosis frompresynaptic terminals.

EXAMPLE 17 Effects on Voltage-Activated Calcium Currents

Presynaptic vesicle release is triggered by an influx of calcium intothe presynaptic terminal. Therefore, Aβ(1-42) globulomer might act onpresynaptic calcium signalling. A common pathway for release of both,glutamatergic and GABAergic vesicles is presynaptic calcium influx viaN-type or P/Q-type calcium channels. Therefore, the effects of Aβ(1-42)on whole-cell calcium currents in cultured hippocampal neurons wereanalyzed. Typical P/Q channel-mediated currents could be reliablyelicited in somatic whole-cell recordings under our culture conditions.In these experiments, 10 mM Ba²⁺ was used as charge carrier in theextracellular solution (see methods). Measurements were performed in thepresence of 10 μM nifedipine (a L-type calcium channel blocker),ω-conotoxin MVIIA (a N-type calcium channel blocker) and blockers ofother voltage-gated ion channels (TTX 0.5 μM, TEA 140 mM, Cs⁺-basedintracellular solution). Rundown of these currents was avoided by adding20 U/ml phosphocreatine kinase and 10 mM tris-phosphocreatine to thepipette solution. Under these conditions, P/Q-type currents were evokedby a depolarizing voltage step to −10 mV (mean amplitude 1015±145 pA;FIG. 21). Aβ(1-42) globulomer reduced the amplitude of these currents to62±7% of control (n=10). This effect was partially reversible in 3/3cells.

If the effect of Aβ(1-42) globulomer on synaptic currents is mediated byblock of P/Q-type calcium channels, it should be mimicked and occludedby the selective P/Q-type calcium channel blocker ω-Agatoxin IVA.Indeed, this toxin (0.5 μM) reduced the frequency of miniature PSCs to27±7% (n=3; amplitude 90±7%), similar to the effect of Aβ(1-42)globulomer. Following pre-incubation with ω-Agatoxin IVA, Aβ(1-42)globulomer had no additional effect on the synaptic currents (n=6,frequency 108±15%; amplitude 102±7% of currents after ω-Agatoxin IVAcontrol; FIG. 23). These data suggest that ω-Agatoxin IVA and Aβ(1-42)globulomer share the same molecular mechanism.

The effect of Aβ(1-42) globulomer on P/Q-type calcium currents wasfurther characterized by steady-state activation and -inactivationprotocols (see methods). Maximal conductance (g_(max)) was 61.7±2.4 nS(control) versus 27.2±3.2 nS (Aβ(1-42) globulomer; p<0.05; n=6; FIG.22). Thus, Aβ(1-42) globulomer reduces the current amplitude withoutaffecting its voltage-dependent activation. In contrast to this markedreduction in conductance (and current amplitude), other kineticparameters were not affected by Aβ(1-42) globulomer. Steady-stateactivation was characterized by V_(h)=−15.4±1.1 mV which was not changedafter application of Aβ(1-42) globulomer (V_(h)=−17.3±1.3 mV; n=6). Theslope of the fitted first-order Boltzmann-equation V_(c) was −7.8±0.3 mVin control solution and −10.8±0.5 mV in Aβ(1-42) globulomer (notdifferent, n=6). Likewise, steady-state inactivation was not affected byAβ(1-42) globulomer, as indicated by stable values for the voltage athalf-maximal inactivation (29.2±0.6 mV in control; 32.4±1.2 mV inAβ(1-42) globulomer; n=4) and for the slope V_(c) (−11.0±0.9 mV versus−12.6±1.1 mV; FIG. 22).). Thus, Aβ(1-42) globulomer reduces the currentamplitude without affecting its voltage-dependent activation orinactivation.

In addition the effects of Aβ(1-42) globulomer on N- and L-type calciumcurrents were analyzed. Besides blockers for Na⁺- and K⁺-channels (seeabove) 0.5 μM ω-agatoxin IVA were added to block P/Q-channels. L-typecalcium currents were isolated by addition of 0.5 μM ω-conotoxin MVIIA.Voltage pulses from −80 mV to −10 mV elicited inward currents of597.7±230.9 pA amplitude which remained stable after addition ofAβ(1-42) globulomer (573.0±225.6 pA; n=3). When N-type currents wereisolated by adding nifedipine (10 μM) instead of ω-conotoxin, the samevoltage clamp protocol elicited inward currents which were, again,insensitive to Aβ(1-42) globulomer (amplitude in control solution1368.9±332.8; amplitude in Aβ(1-42) globulomer 1399.8±376.4 pA; n=3).When all blockers were added together, the remaining calcium current(possibly R-type) was too small for a detailed analysis (<100 pA),indicating that this component was only marginally expressed in thecultured hippocampal neurons.

EXAMPLE 18 Rescue by Roscovitine

Application of roscovitine in the presence of Aβ(1-42) globulomer didindeed partially recover the frequency of synaptic currents. While inthese experiments Aβ(1-42) globulomer reduced the frequency ofspontaneous PSCs to 38±10% of control, application of roscovitine (20μM) brought this parameter back to 75±13% (n=5; FIG. 23).

Together, these data indicate that Aβ(1-42) globulomer reduces thefrequency of spontaneous and miniature synaptic currents by suppressionof presynaptic calcium influx via P/Q-type calcium channels.

THIRD SERIES OF EXPERIMENTS EXAMPLE 19 Rescue of Spontaneous SynapticActivity by Isoproterenol

Isoproterenol was used at a final concentration of 15 μM, by adding itsimultaneously with Aβ(1-42) globulomer (final concentration of Aβglobulomer corresponding to approximately 1 μM of Aβ monomer).Isoproterenol is known (Huang C.-C., et al., The Journal ofNeuroscience, 1996, 16(3): 1026-1033, Huang C.-C., et al., The Journalof Neuroscience, 1998, 18(6): 2276-2282) to increase the Ca²⁺ influxthrough the P/Q type voltage-gated presynaptic calcium channel.

Application of isoproterenol in the presence of Aβ(1-42) globulomer didindeed recover the frequency of synaptic currents. While in theseexperiments Aβ(1-42) globulomer reduced the frequency of spontaneousIPSCs to 57±7% of control, application of isoproterenol (15 μM) broughtthis parameter back to 122±58% (n=6; FIG. 24).

This demonstrates that the P/Q type voltage-gated presynaptic calciumchannel activator isoproterenol is capable of restoring the frequency ofspontaneous synaptic events under the influence of Aβ globulomer to thatof untreated cells, i. e., that the P/Q activator may be used to reversethe detrimental effects of Aβ globulomer.

EXAMPLE 20 Enhancing P/Q Calcium Currents by RoscovitinePrevents/Reverses Chronic Aβ Globulomer-Induced Deficits on EvokedSynaptic Transmission in Hippocampal Tissue

Rat hippocampal slice cultures (9 days old Wistar rats; 15-17 DIV) wereincubated over night with either Aβ(1-42) globulomer (at a concentrationcorresponding to approximately 1 μM of Aβ monomer), Aβ(1-42) globulomer(at a concentration corresponding to approximately 1 μM of Aβmonomer)+20 μM roscovitine, or control (SDS). Recordings were performed(in artificial cerebrospinal fluid) from CA1 stratum radiatum afterstimulation of the Schaffer collateral at different intensities.

Results are shown in FIG. 25, demonstrating that the application ofglobulomer strongly suppresses synaptic transmission. Co-application of20 μM roscivitine completely prevents/reverses the globulomer-induceddeficit.

EXAMPLE 21 Enhancing P/Q Calcium Currents by Roscovitine Analogue APrevents/Reverses Chronic Aβ Globulomer-Induced Deficits on EvokedSynaptic Transmission in Hippocampal Tissue

Rat hippocampal slice cultures (11 days old Wistar rats; 23-24 DIV) wereincubated over night with either Aβ(1-42) globulomer (at a concentrationcorresponding to approximately 1 μM of Aβ monomer), Aβ(1-42) globulomer(at a concentration corresponding to approximately 1 μM of Aβmonomer)+20 μM roscovitine analogue A, or control (SDS). Recordings wereperformed (in artificial cerebrospinal fluid) from CA1 stratum radiatumafter stimulation of the Schaffer collateral at different intensities.

Results are shown in FIG. 26, demonstrating that the application ofglobulomer strongly suppresses synaptic transmission. Co-application of20 μM roscivitine analogue A completely prevents/reverses theglobulomer-induced deficit.

EXAMPLE 22 Effect of Extracellular Ca²⁺ on sPSC Frequency AfterTreatment with Aβ(1-42) Globulomer

Spontaneous synaptic activity was measured in cultured hippocampalneurons using whole-cell voltage clamp techniques (V_(hold)=−70 mV).Under the ionic conditions used (E_(Cl)˜−10 mV) all synaptic eventsappeared as inward currents.

The effects of Aβ(1-42) globulomer (at a concentration corresponding toapproximately 1 μM of Aβ monomer) were assessed by comparingspontaneously occurring postsynaptic currents (sPSCs) in single cells in5 min intervals in the presence or absence of globulomer in bathsolution containing 1 mM Ca²⁺. Currents recorded prior to the additionof the globulomer served as control describing basal synaptictransmission. Currents recorded in the interval immediately afterapplication were analysed with respect to the control data. Afterwards,extracellular Ca²⁺ was elevated from 1 mM to 4 mM (leaving theconcentration of globulomer unchanged). Currents in the following 5 minrecording interval were again analysed with respect to control data.

Basal frequency of sPSCs in 1 mM Ca²⁺ was 4.2±1.2 Hz. Bath-applicationof Aβ(1-42) globulomer rapidly reduced the sPSC frequency to 63±7% ofcontrol (p<0.05; n=6; FIG. 27 A+B). After elevation of extracellularCa²⁺ to 4 mM, sPSC frequency partially recovered to 77±13% of control(FIG. 27 B). In 4 of 6 cells tested, sPSC frequency increased, whereasit remained unaltered in the other 2 cells (FIG. 27 C).

Median amplitude of sPSCs under control conditions was 27.7±2.2 pA andremained unaltered after addition of Aβ(1-42) globulomer (97±5%; FIG. 27D) or subsequent elevation of extracellular Ca²⁺ (98±6%).

In most cases, prominent currents with amplitudes up to 2000 pA occurreddirectly after elevation of extracellular Ca²⁺-concentration. Thesecurrents with multiple peaks (see FIG. 27 E) were rejected fromanalysis.

This clearly demonstrates that the principle of activating the P/Q typepresynaptic calcium channel is effective in compensating the detrimentaleffects exerted by Aβ globulomer.

1. A method for treating amyloidosis active, the method comprisingadministering an agonist of the P/Q type voltage-gated presynapticcalcium channel, to a subject in need there of.
 2. The method of claim1, wherein the amyloidosis is Alzheimer's disease or Down's Syndrome. 3.The method of claim 1, wherein the agonist binds to the P/Q typevoltage-gated presynaptic calcium channel.
 4. The method of claim 1,wherein the agonist affects the P/Q type voltage-gated presynapticcalcium channel with an EC50 of less than 120 μM.
 5. The method of claim1, wherein the agonist affects the P/Q type voltage-gated presynapticcalcium channel with an EC50 that is lower than the EC50 with which itaffects the N and/or R type presynaptic calcium channel.
 6. The methodof claim 1, wherein the agonist affects the N and/or R type presynapticcalcium channel with an EC50 of more than 54 μM.
 7. (canceled)
 8. Themethod of claim 1, wherein the treatment is for the restoration ofsynaptic function and/or plasticity.
 9. The method of claim 1, whereinthe treatment is for the restoration of long-term potentiation.
 10. Themethod of claim 1, wherein the treatment is for the restoration ofmemory function.
 11. The method of claim 1, wherein the treatment is forthe restoration of performance of activities of daily living (ADL)capacity in the subject.
 12. The method of claim 1, wherein the agonistis a compound having formula selected from the group consisting of:

and a pharmaceutically acceptable salt thereof, wherein if the compoundhas formula (Ia), then R¹ is hydrogen or C₁-C₆ alkyl; R^(2a), R^(2b) areindependently hydrogen, C₁-C₆-alkyl, C₂-C₆-alkenyl, C₃-C₈-cycloalkyl,optionally substituted C₆-C₁₂-aryl or optionally substitutedC₆-C₁₂-aryl-C₁-C₄-alkyl, or R^(2a), R^(2b) together are C₂-C₅-alkylene;Q is NR³; R³ is hydrogen, C₁-C₆-alkyl or optionally substitutedC₆-C₁₂-aryl; X is N or CR⁴; R⁴ is hydrogen or C₁-C₆-alkyl, Y is N orCR⁵; and R⁵ is hydrogen or C₁-C₆-alkyl; wherein if the compound hasformula (Ib), then R¹ hydrogen or C₁-C₆ alkyl; R^(2a), R^(2b) areindependently hydrogen, C₁-C₆-alkyl, C₂-C₆-alkenyl, C₃-C₈-cycloalkyl,optionally substituted C₆-C₁₂-aryl or optionally substitutedC₆-C₁₂-aryl-C₁-C₄-alkyl, or R^(2a), R^(2b) together are C₂-C₅-alkylene;Q is NR³; R³ is hydrogen, C₁-C₆-alkyl or optionally substitutedC₆-C₁₂-aryl; X is N or CR⁴; R⁴ is hydrogen or C₁-C₆alkyl, Y is N or CR⁵;and R⁵ is hydrogen or C₁-C₆-alkyl; and wherein if the compound hasformula (II), then R¹ is C₁-C₆-alkyl or C₃-C₈cycloalkyl; R^(2a), R^(2b),R^(2c), R^(2d), R^(2e) are independently hydrogen, halogen, C₁-C₄-alkyl,optionally substituted phenyl, OH, SH, CN, CF₃, O—CF₃, C₁-C₄-alkoxy;NH₂, NH—C₁-C₄-alkyl, alkyl)₂, or R^(2b) and R^(2c) or R^(2c) and R^(2d)together with the carbon atoms to which they are attached form anoptionally substituted anellated C₅-C₇ carbocyclic ring; and thepharmacologically useful salts thereof.